Open-Chamber Co-Culture Microdevices for Single-Cell Analysis of Skeletal Muscle Myotubes and Motor Neurons with Neuromuscular Junctions

  • Yamaoka Nao
  • Kazunori ShimizuEmail author
  • Imaizumi Yu
  • Ito Takuji
  • Okada Yohei
  • Honda Hiroyuki
Original Article


Degeneration of motor neurons and skeletal muscles or the collapse of neuromuscular junctions (NMJs) causes progressive motility disturbances in many neuromuscular diseases. Although various microdevices for the co-culture of skeletal muscle myotubes and motor neurons have been developed to investigate neuromuscular diseases in vitro, it remains difficult to isolate single myotubes and motor neurons from the device for single-cell analyses, such as gene expression analysis. Here, we developed open chamber-coculture microdevices that contain cell culture chambers with narrow widths. Given the small chamber width (0.2 mm), the device significantly prevented the overlap among myotubes within the chamber. The percentage of non-overlapping was 95.6 ± 7.7% for the 0.2-mmwidth chamber and 11.8 ± 6.4% for the 7-mm-width chamber as a control. In addition, the device with the 0.2-mm chamber promoted myotube maturation, as indicated by the longer widths and lengths of the myotubes relative to those in the control chamber. Single C2C12 myotubes and human induced pluripotent stem cell (hiPSC)-derived motor neurons were successfully collected from the device with the 0.2-mm chamber using a micromanipulator equipped with a glass capillary. Furthermore, myotubes and hiPSC-derived motor neurons were co-cultured in the device with the 0.2- mm chamber, and the formation of NMJs were observed. Thus, the developed device is a useful tool for performing single-cell analysis for studying neuromuscular diseases in vitro.


Neuromuscular junction Neuromuscular disease Co-culture Microdevice Human induced pluripotent stem cells 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Witzemann, V. Development of the neuromuscular junction. Cell Tissue Res. 326, 263–271 (2006).CrossRefGoogle Scholar
  2. 2.
    Darabid, H., Perez-Gonzalez, A.P. and Robitaille, R. Neuromuscular synaptogenesis: coordinating partners with multiple functions. Nat. Rev. Neurosci. 15, 703–718 (2014).CrossRefGoogle Scholar
  3. 3.
    Fischer, L.R., Culver, D.G., Tennant, P., Davis, A. A., Wang, M.S., Castellano-Sanchez, A., Khan, J., Polak, M.A. and Glass, J.D. Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp. Neurol. 185, 232–240 (2004).CrossRefGoogle Scholar
  4. 4.
    Wong, M. and Martin, L.J. Skeletal muscle-restricted expression of human SOD1 causes motor neuron degeneration in transgenic mice. Hum. Mol. Genet. 19, 2284–2302 (2010).CrossRefGoogle Scholar
  5. 5.
    Robitaille, R., Garcia, M.L., Kaczorowski, G.J. and Charlton, M.P. Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release. Neuron 11, 645–655 (1993).CrossRefGoogle Scholar
  6. 6.
    Gurney, M., Pu, H., Chiu, A., Dal Canto, M., Polchow, C., Alexander, D., Caliendo, J., Hentati, A., Kwon, Y., Deng, H. and et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Sciences (N. Y.) 264, 1772–1775 (1994).CrossRefGoogle Scholar
  7. 7.
    He, X.P., Yang, F., Xie, Z.P. and Lu, B. Intracellular Ca2+ and Ca2+/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3. J. Cell Biol. 149, 783–791 (2000).CrossRefGoogle Scholar
  8. 8.
    Guo, X.F., Gonzalez, M., Stancescu, M., Vandenburgh, H.H. and Hickman, J.J. Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system. Biomaterials 32, 9602–9611 (2011).CrossRefGoogle Scholar
  9. 9.
    Das, M., Rumsey, J.W., Bhargava, N., Stancescu, M. and Hickman, J.J. A defined long-term in vitro tissue engineered model of neuromuscular junctions. Biomaterials 31, 4880–4888 (2010).CrossRefGoogle Scholar
  10. 10.
    Ionescu, A., Zahavi, E.E., Gradus, T., Ben-Yaakov, K. and Perlson, E. Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance. Eur. J. Cell Biol. 95, 69–88 (2016).CrossRefGoogle Scholar
  11. 11.
    Tong, Z., Seira, O., Casas, C., Reginensi, D., Homs-Corbera, A., Samitier, J. and Antonio Del Rio, J. Engineering a functional neuro-muscular junction model in a chip. RSC Adv. 4, 54788–54797 (2014).CrossRefGoogle Scholar
  12. 12.
    Shimizu, K., Genma, R., Gotou, Y., Nagasaka, S. and Honda, H. Three-Dimensional Culture Model of Skeletal Muscle Tissue with Atrophy Induced by Dexamethasone. Bioengineering 4, (2017).Google Scholar
  13. 13.
    Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. and Yamanaka, S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).CrossRefGoogle Scholar
  14. 14.
    Nagashima, T., Shimizu, K., Matsumoto, R. and Honda, H. Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine. Sci. Rep. 8, 12427 (2018).CrossRefGoogle Scholar
  15. 15.
    Shimojo, D., Onodera, K., Doi-Torii, Y., Ishihara, Y., Hattori, C., Miwa, Y., Tanaka, S., Okada, R., Ohyama, M., Shoji, M., Nakanishi, A., Doyu, M., Okano, H. and Okada, Y. Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells. Mol. Brain 8, 79 (2015).CrossRefGoogle Scholar
  16. 16.
    Arai, S., Okochi, M., Shimizu, K., Hanai, T. and Honda, H. A single cell culture system using lectin-conjugated magnetite nanoparticles and magnetic force to screen mutant cyanobacteria. Biotechnol. Bioeng. 113, 112–119 (2016).CrossRefGoogle Scholar
  17. 17.
    Shimizu, K., Fujita, H. and Nagamori, E. Micropatterning of single myotubes on a thermoresponsive culture surface using elastic stencil membranes for single-cell analysis. J. Biosci. Bioeng. 109, 174–178 (2010).CrossRefGoogle Scholar
  18. 18.
    Velleman, S.G. and McFarland, D.C. Myotube morphology, and expression and distribution of collagen type I during normal and low score normal avian satellite cell myogenesis. Dev., Growth Differ. 41, 153–161 (1999).CrossRefGoogle Scholar
  19. 19.
    Bettadapur, A., Suh, G.C., Geisse, N.A., Wang, E.R., Hua, C., Huber, H.A., Viscio, A.A., Kim, J.Y., Strickland, J.B. and McCain, M.L. Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels. Sci. Rep. 6, 28855 (2016).CrossRefGoogle Scholar
  20. 20.
    Ostrovidov, S., Ahadian, S., Ramon-Azcon, J., Hosseini, V., Fujie, T., Parthiban, S.P., Shiku, H., Matsue, T., Kaji, H., Ramalingam, M., Bae, H. and Khademhosseini, A. Three-dimensional co-culture of C2C12/PC12 cells improves skeletal muscle tissue formation and function. J. Tissue Eng. Regener. Med. 11, 582–595 (2017).CrossRefGoogle Scholar

Copyright information

© The Korean BioChip Society and Springer 2018

Authors and Affiliations

  • Yamaoka Nao
    • 1
  • Kazunori Shimizu
    • 1
    Email author
  • Imaizumi Yu
    • 1
  • Ito Takuji
    • 2
  • Okada Yohei
    • 2
  • Honda Hiroyuki
    • 1
    • 3
  1. 1.Department of Biomolecular Engineering, Graduate School of EngineeringNagoya UniversityNagoyaJapan
  2. 2.Department of NeurologyAichi Medical University School of MedicineAichiJapan
  3. 3.Innovative Research Center for Preventive Medical EngineeringNagoya University, Furo-cho, Chikusa-kuNagoyaJapan

Personalised recommendations